Effect of some biocontrol agents against root-knot nematode (Meloidogyne incognita race2)

Culture filtrate of four rhizospheric fungi and four biocontrol agents were studied in vitro for their efficacy against Meloidogyne incognita race 2. The per cent mortality and egg hatching inhibition was proportional to the concentration of culture filtrate and the duration of exposure period. Culture filtrates of Trichoderma viride, Trichoderma harzianum, Trichoderma sp., Fusarium sp., Penicillium sp. and Aspergillus sp. significantly induced inhibition of egg hatching and mortality of Meloidogyne incognita race 2.The highest percentage of inhibition of egg hatching and juvenile mortality was recorded in Trichoderma harzianum followed by Trichoderma viride and Trichoderma sp.


INTRODUCTION
Root-knot nematode (Meloidogyne spp.) is an important plant pathogen affecting crop production throughout the world. Since, indiscriminate use of nematicides is responsible for environmental and human health concerns; the search for new microbial strains as nematode control agents is relevant. As fungi cohabit together with nematodes in the rhizosphere, their to xic metabolites may be responsible for keeping a low level of nematode populations [1]. The search for nematotoxic or antagonistic compounds in culture filtrates has greatly intensified in recent years, due to the number of to xins, enzy mes or co mpounds derivable fro m their metabolites [2][3][4][5][6][7]. Assays with culture filtrates may provide first informat ion about the role of a fungus in the p lant rh izosphere, as in vitro studies showed toxic and inhib itory effects of several filtrates toward plant parasitic nematodes [8]. To xic effects of fungal culture filtrates on M. incognita have been studied by several workers [9-16] and had showed different levels of efficacy [17][18][19][20]. Due to the differences of soil ecological types and climate, a broad range of fungi remains far unexp lored. Therefore, p resent study was made to isolate rh izospheric fungal associations of root knot nematode infected plants and evaluate the potential of some isolated fungi and already recognized biocontrol agents (against insect pests and diseases) on hatching of eggs and mortality of secondstage juveniles of Meloidogyne incognita race 2 in vitro.

II. MATERIALS AND METHODS Collection of samples
Soil samples were collected in different localities around Jorhat, Assam comp rising an area appro ximately 1000 ha, in order to identify the root-knot nematode infection. To isolate the fungal antagonists fro m rhizosphere soils of infested cucurbits, tomato, brinjal, o kra, cabbage, citrus, banana and tea, a total of 100 soil (500 g each) were collected. Samp les were stored at 15 0 C for not more than one week. Fungal isolation and identification Soil mycoflora was isolated by serial d ilution pour plate technique [21,22]. One g of rhizosphere soil was dispensed in 9 ml sterile water, fro m the 10 -5 dilution, 50 μl were inoculated over Petri plates containing PDA media. The plates were incubated at room temperature 24±2°C for 48 hrs. Materials of the pure culture were mounted in Lactophenol, stained with Cotton blue and the morphological observations of hyphae, sporangiophore/conidiophores and conidia were done with the help of a Co mpound light microscope at 400X magnificat ion [23,24]. A xenic cultures of the fungi were obtained by single spore isolations [25] and the cultures were maintained on PDA slants. Trichoderma viride, Trichoderma harzianum, Beauveria bassiana, Metarhizium anisopliae were procured from the Depart ment of Plant Pathology, AAU, Jorhat, Assam.

Nematode inoculum and mass culturing
The inoculu m o f root-knot nematode M. incognita race 2 was collected fro m naturally infested tomato crop in field and single egg mass was used to raise pure culture. Mass culturing of nematodes was done on tomato variety Sel 7, in order to get regular supply of the inoculums for the experiment. One month old tomato seedlings were inoculated with small volu me of egg suspension approximately consisting of 2000 eggs of M.incognita race 2. These pots were watered and kept in glasshouse at temperature 28-35°C.

Preparation of fungal culture filtrates
To evaluate the nematicidal potential of the cell free fungal culture filtrate the most frequently occurring isolates belonging to the genera of Trichoderma, Aspergillus, Penicillium and Fusarium were selected. Beauveria bassiana, Metarhizium anisopliae, Trichoderma viride and Trichoderma harzianum were procured fro m the Depart ment of Plant Pathology, AAU, Jorhat, Assam. These strains were inoculated on to Petri p lates containing Potato Dextrose Agar mediu m and incubated for 7 to 10 days at 27°C. Fro m these actively g rowing cultures, one disc each of 0.5 cm d iameter was transferred to 250 mL Erlen meyer flask containing 50 mL Potato Dextrose broth. These flasks were incubated at 27±1°C fo r 15 days. The culture was filtered through two layers of Whatman filter Paper No.1. Filtrates thus obtained were designated as standard solution (100%). Different dilutions (50%, 25%, and 10%) of each fungal filtrate were prepared by adding required amount of sterilized distilled water.

Hatching test
To determine the effect of culture filtrate on the hatching of eggs of M. incognita sterilized Petri dishes of 5 cm dia were separately pipette two ml of culture filt rate. Five sterilized healthy egg masses of nearly uniform size o f M.incognita were transferred to each dish. The egg masses placed in culture mediu m served as control. All Petri dishes were kept at 28±2°C in co mpletely rando mized design, replicated thrice. Observations were recorded on every 24 h interval up to 72 h with the aid of stereomicroscope. The per cent egg hatch was calculated by the following formula and mean of three replications was presented in Table.2.

No. of hatched juveniles
No. of hatched+ unhatched eggs

Mortality of second stage juveniles (J2)
For determining the effect of fungal filtrates on juvenile mortality of M.incognita race 2, egg masses were collected fro m an infested root and allowed to hatch in d istilled water with aeration. The hatched J2 were collected in a beaker. One hundred freshly hatched second stage juveniles were transferred to 5 cm dia Petri dishes containing 2 ml filtrates of different dilutions of each fungus and mediu m separately. Equal number of J2 was also transferred to separate Petri dishes containing culture mediu m to serve as control. Petri dishes were kept at 28±2°C temperature in comp letely randomized design, replicated thrice. Observation on the number of dead J2 for every 24, 48 and 72 h of exposure was recorded with the aid of stereomicroscope and per cent mortality of juveniles was calcu lated. The J2 were considered dead when they did not move when probing with a fine needle Mean percentage of dead J2 was estimated using the following formula and presented in the Table 3. Total number of dead juveniles Per cent mortality= ----------------------x100 Total number of juveniles STATISTICAL ANALYSIS Per cent egg hatch and per cent mortality data was subjected to statistical analysis using the three factorial co mp letely randomized design statistical package. The critical differences in main effects i.e. isolates, concentration, and time of exposure as well as in their interactions were tested at P=0.05.

III. RESULTS
A total of four isolates of different genera of fungi were isolated fro m the soil rhizosphere of M.incognita race 2 infected plants. Trichoderma sp. Fusarium sp., Aspergillus sp. and Penicillium sp. was isolated fro m rh izospheric soil of banana, cowpea, brinjal and cucumber respectively. The results presented in Table 1 revealed significant differences among isolates (biocontrol agent) (T), concentration of culture filtrate(C) and exposure period(t). The culture filt rate of Trichoderma harzianum followed by T.viride, T.sp. Aspergillus sp., P. sp. and F.sp. adversely affected the larval hatching of M.incognita race 2. Irrespective of concentration of culture filt rate (C) and time of exposure period (t), T.harzianum was the most effective bioagent followed by T.viride and T.sp. as the hatching of M.incognita was suppressed. Similarly, irrespective of isolate (biocontrol agent) (T) and concentration of culture filtrate (C), time of exposure(t) also affected the larval hatching. With increase in exposure period up to 72 hours there was a correspondingly increased in egg hatching. With increase in the dilution of culture filtrate, the cumu lative hatching was increased irrespective of isolate (T) and time of exposure period (t). Highest inhibition in hatching was obtained in 100% concentration of each fungal culture filtrates. The percentage hatching of M.incognita was 18.35% during 72 h exposure in the 100% concentrations of culture filtrates of Trichoderma viride followed by T.harzianum with percentage hatching 20.36%. Beauveria bassiana and Metarhizium anisopliae showed negligib le effect on inhibit ion of egg hatching of M.incognita. Hatching percentage was 59.34%, 58.35% respectively at 10% concentration of culture filtrate during 72 h exposure period. The data showed in the Table 2 revealed that all the culture filtrates of isolates were having nemat icidal effect of varying degree on M.incognita race 2. Per cent mortality of nematodes was directly proportional to the concentration of culture filt rate and the period of exposure. Irrespective of concentration of culture filtrate (C) and duration of exposure(t), six isolates namely Trichoderma harzianum, Trichoderma viride, Trichoderma sp., Fusarium sp., Aspergillus sp. and Penicillum sp. were exh ibited nematicidal effects on M. incognita J2.

IV.
DISCUSSION Culture filtrates of many fungi possess nematicidal activity against nematodes, due to the production of toxic metabolites [26].Variable effect of fungal filt rates on hatching and mortality of root-knot nematode M.incognita race 2 observed in the present study may be attributed to the varied nature of toxic metabolites produced by different fungi. Species of Trichoderma, Fusarium, Paecilomyces, Aspergillus, Penicillium are known to produce toxins and antibiotics like viridian, fusaric acid, lilacin, o xalic acid and penicillic acid [27,28]. Various mechanis ms have been suggested for the biocontrol activity o f Trichoderma spp. against phytopathogenic fungi: antibiosis, co mpetition, mycoparasitism, and en zy matic hydrolysis [29,30]. Trichoderma spp. is utilized in the production of a nu mber of antibiotics, such as trichoderin, trichodermol A and harzianolide. Trichoderma produces molecules such as 6pentyl α-pyrone, VOCs and enzy mes [31] that can attack the cuticle o f nematodes. Also, its hyphae form a physical barrier, wh ich is a difficult step for nematodes, since the fungus grows along with the plant roots. Successful bassiana may have more than a single bioactive metabolite that are responsible for nematicidal act ivities, and each metabolite may act on a different site. Ghayedi and Abdollahi [48] purified the isolated fungus and also they showed the biocontrol potential of the isolate on Heterodera avenae, with 47.1% of larval mortality. Chen et al., [49] found that B. bassiana showed little parasitism of nematode eggs but reduced hatch of Heterodera glycines. Studies have shown that Beauveria can produce beauvericin and oosporin. Beauvericin has a weak activ ity against M. incognita [50][51][52][53]. The percentage mortality and inhibition of hatching of root-knot nematode were directly proportional to the concentration of culture filt rates of B.bassiana [54]. Biocontrol potential of M. anisopliae against some species of root knot nematodes has been shown [55][56][57][58]. The lethal effect o f M. anisopliae culture extract has been also reported [59].So me species of Metarhizium has root colonization ability [60,61]. So me isolates of M. anisopliae have endophytic behavior [62]. The fungus produces sticky conidia that attach to nematode cuticle [63]. The conidia germinate, parasitize and kill the cadaver, by direct penetration and producing the infective hyphae inside the nematode body. The fungus produces some cyclopeptides and destruxins which may play an important role in its pathogenicity [64]. Prio r to any d irect attack to the host, the fungus produces destruxin A and destruxin B that can kill the host [65].